1. Field of the Invention
The present invention relates to catalytic antibodies, and more particularly to catalytic antibodies and a method for producing catalytic antibodies which are capable of hydrolyzing phosphoester bonds in a metal dependent manner.
2. Description of the State of Art
Chemical reactions in biological systems rarely occur in the absence of a catalyst. These catalysts, referred to as enzymes, are highly specific in the reactions they catalyze and the substrates utilized, and while they may go through temporary changes they are not consumed in the reaction. Enzymes accelerate reactions by factors of at least a million. Indeed, most reactions in biological systems do not occur at perceptible rates in the absence of enzymes. For example, it has been estimated that the phosphodiester bonds in DNA have a half-life for hydrolytic cleavage of 200 million years. In contrast, many DNases (enzymes that catalyze this reaction) can hydrolyze the phosphodiester bonds in DNA in a matter of seconds.
An enzyme, as a catalyst, cannot alter the equilibrium of a chemical reaction. This means that an enzyme accelerates the forward and reverse reaction by precisely the same factor. Consider the interconversion of A and B. Suppose that in the absence of enzyme the forward rate (k.sub.F) is 10.sup.-3 sec.sup.-1 and the reverse rate (k.sub.R) is 10.sup.-5 sec.sup.-1. The equilibrium constant K is given by the ratio of these rates. ##EQU1## The equilibrium concentration of B is 100 times that of A, whether or not enzyme is present. However, it will take several hours to approach this equilibrium without enzyme, whereas equilibrium would be attained within a second when enzyme is present. Thus, enzymes accelerate the attainment of equilibria but do not shift their positions.
A chemical reaction, A.revreaction.B, goes through a transition state that has a higher energy than either A or B. The rate of the forward reaction depends on the temperature and on the difference in free energy between that of A and the transition state, which is referred to as the Gibbs free energy of activation and symbolized .DELTA.G.sup..dagger-dbl., as shown in FIG. 1a. EQU .DELTA.G.sup..dagger-dbl. =G.sub.transition state -G.sub.substrate
Enzymes accelerate reactions by decreasing .DELTA.G.sup..dagger-dbl., the activation barrier. The combination of substrate and enzyme at a specific region of the enzyme called the active site, creates a new reaction pathway whose transition state energy is lower than it would be if the reaction were taking place in the absence of enzyme as shown in FIG. 1b.
The design and generation of efficient catalysts with any desired specificity is a "holy grail" for chemists and biochemists alike. Chemists have concentrated on the use of nature's most efficient catalysts, the enzymes, in the field of organic synthesis (see, Wong, C. H., et al., Curr. Opinion. Struc. Biol., 8:694 (1993)), "host-guest" interactions or supramolecular chemistry, and in the use of small, reactive organic molecules as models of enzymes. Likewise, biochemists have used molecular biology as well as chemical techniques to modify existing biological molecules. Recently chemistry and biochemistry have met in the development of catalytic antibodies. Here, an understanding of chemical reactivity has been combined with the diversity of the immune system to generate antibodies capable of not only binding to their antigens, but effecting chemical reactions upon them. Since the first reports of catalytic antibodies in 1986, over fifty different reactions have been shown to be antibody catalyzed (Lerner, R. A., et al., Science, 252:659 (1991)). However, no metal dependent catalytic antibodies currently exist that can hydrolyze phosphate ester bonds, and while catalysts for hydrolyzing phosphate esters have representatives in many of the fields mentioned previously, they lack the specificity to which catalytic antibodies would lend themselves.
Antibodies or immunoglobulins (Ig's) are large proteins that consist of four polypeptide chains: two identical light (L) chain polypeptides and two identical heavy (H) chain polypeptides held together by disulfide bridges and non-covalent bonds. The four chains contain defined Variable (V), Diversity (D) (heavy chain only), Joining (J) and Constant (C) regions. The DNA and amino acid sequence of the C region is relatively conserved within a given animal species while the V region sequence is antigen-dependent. Pairing of the heavy and light chain V regions creates an antigen-binding site (paratope) which recognizes a single antigenic determinant (epitope). Within each variable region are three complementarily-determining regions (CDRs) of extremely high variability which provide the basis for the diversity of the antibody molecule. The specificity of antibodies for their antigens can exceed that of enzymes for substrates. Antibodies bind antigens or haptens with association constants that range from 10.sup.4 to 10.sup.14 M.sup.-1. Small antigens are typically bound in a cleft, but for larger molecules the binding site can be an extended surface that can cover 600 to 800 .ANG..sup.2.
While the genetic mechanism whereby an antibody gene forms has been estimated to be capable of producing over 10.sup.11 different antibody molecules for an individual, the range of reactions that can be catalyzed by enzymes composed of only the 20 natural amino acids fall far short of this number. Enzymes, however, may utilize the existence of nonpeptidyl catalytic auxiliaries, referred to as cofactors, to greatly expand the range of reactions that can be catalyzed. These cofactors include metal ions, hemes, thiamine, flavins, and pyridoxal phosphate.
Metal ions have long been recognized as essential components of living systems, and strategies that would allow incorporation of metal ions into antibody combining sites should, by analogy to enzymes, expand the scope of antibody catalysis. In this case, metal ions may play a number of roles. One would be the ability to orient the substrate correctly in the active site, serving as a template by neutralizing anionic charges on the substrate. A second would be to act as a super Lewis acid, activating the substrate by withdrawing electrons from the substrate, making it more susceptible to nucleophilic attack. Another role would be to coordinate the attaching water molecule in a manner that greatly reduces its pK.sub.a and aids the delivery of a hydroxide ion nucleophile at physiological pH.
There have been some attempts at engineering metal binding antibodies. For example, Sarvetnick, N., et al., disclosed their attempt to create a metal-binding antibody which involved producing transgenic mice with a metal ion-binding light chain in the genome. The light chain has a three-histidine site with specificity for Cu(II) and Zn(II). These transgenic mice were immunized with a fluorescein conjugate. The three-histidine light chain was found in two of six hybridomas isolated. While this work is encouraging with regards to expanding the chemical potential of the immune system, there are, however, some concerns. The authors did not show that metal ions actually bound to the isolated antibodies, and furthermore, this work did not demonstrate that a metal ion and the fluorescein antigen bind simultaneously, or in a geometry that allows for a chemical reaction (Sarvetnick, N., et al., Proceedings of the National Academy of Sciences., 90:4008 (1993)).
A more systematic approach involved engineering the three-histidine site into a light chain variant of the same antibody, disclosed by Wade, W. S., et al. Four sites were modified and it was shown that all mutant antibodies bound fluorescein. Based on tryptophan fluorescence quenching, two of the four sites exhibited metal affinities consistent with complexation by three ligands. The specificity of the tightest binding site was probed by mutagenesis. Here, the second highest isolated affinity site showed a metal-dependent increase in fluorescein binding, which indicates a ternary complex. Several combinations of modifications having only four amino acid changes gave affinities in a potentially useful range for antibody catalysis (Wade, W. S., et al., J. Am. Chem. Soc., 115:4449 (1993)).
As an alternate approach, Pessi and coworkers have generated what they call the "minibody". This molecule was constructed by incorporating the three-histidine metal-binding site into the immunoglobulin heavy chain variable domain. The resulting molecule had a novel .beta.-sheet scaffold and two regions corresponding to hypervariable loops. The protein was folded, compact and bound metal ions (Pessi, A., et al., Nature, 362:367 (1993)).
The alternative to engineering metal-binding antibodies as discussed above has focused solely on inducing antibodies to transition state analogues as haptens. For example, Lerner and coworkers were the first to use cofactor containing haptens successfully for the induction of catalytic antibodies capable of cofactor-assisted peptide bond cleavage. Antibodies were made against a covalent Co.sup.3+ N.sub.4 ! compound that mimicked the transition state of a cofactor-assisted peptide bond cleavage. This work, however encouraging, was preliminary, as no kinetic constants were presented, and further work has not appeared (Lerner, R. A., et al., Proceedings of the National Academy of Sciences, 90:6385-6389 (1993)). Another example of cofactors in catalytic antibodies is an antibody-catalyzed porphyrin metallation. Ferrochelatase is an enzyme that catalyzes the insertion of Fe.sup.2+ into protoporphyrin. N-alkylated porphyrins have a distorted macrocycle and are thought to closely resemble the transition state for the chelation of the porphyrin. Antibodies were generated against the putative transition state analogue N-methyl-mesoporphyrin IX; however, no metal ion was present in the hapten. Also, the authors reported that binding of metal ions by the antibody was not saturable, and did not contribute to catalysis in any significant way (Schultz, P. G., et al., Science 249:781-783 (1990)).
The most recent example of a catalytic antibody utilizing cofactors is one where the antibody was not generated to metal ions or metal ions complexes. A rationally designed hapten with structural features that could translate into induction of antibodies with a metal binding pocket was used. It was determined that one of the antibodies utilized a substrate with a pyridine moiety only when it was complexed with Zn.sup.2+. However, no antibodies with metal ion binding sites were obtained (Lerner, R. A., et al., J. Am. Chem. Soc., 115:4906-4907 (1993)).
To date, research in the field of metal dependent catalytic antibody induction is based entirely on using transition state analogues as haptens. This approach to generating catalytic antibodies however is problematic for the hydrolysis of phosphodiesters. The transition state for phosphodiester bond hydrolysis is trigonal pyramidal; that is, 5-coordinate. The classical approach to generating catalytic antibodies for phosphodiester bond hydrolysis would be to synthesize a suitably stable 5-coordinate compound for use as a hapten and screen the resulting antibodies for catalytic activity. Unfortunately, phosphorus does not form stable 5-coordinate complexes that resemble this transition state. Other elements, such as vanadium (V), with this geometry are too unstable in aqueous solutions and would be hydrolyzed before an immune response could be mounted. Currently there is no known catalytic antibody that can hydrolyze phosphodiester bonds, nor are there any known catalytic antibodies that can independently bind a metal ion that acts as a cofactor in a chemical reaction.
There is still a need, therefore, for catalytic antibodies and a method for producing catalytic antibodies that are capable of hydrolyzing phosphodiester bonds in a metal dependent manner.